Alternative splicing (AS) of precursor messenger RNA molecules can generate immense complexity from just a single gene (1). Intriguingly, AS is greatest in complex tissues such as the human brain, which may explain why several diseases, are caused by defects in RNA-binding proteins that control AS (2). Thus, AS is strongly related to biological complexity and the proper function of complex cell types, like neurons. The most extreme example that demonstrates this relationship is the Down syndrome cell adhesion molecule (Dscam) of Drosophila melanogaster. Dscam can be alternatively splicing to conceivably generate over 38,000 different protein isoforms; without this complexity, neurons do not wire properly (3). The aim of this proposal is to understand the mechanisms by which Dscam AS is controlled in Drosophila neurons. Preliminary results suggest that the expression of particular Dscam isoforms may be highly biased for specific neurons. How this might occur is not known, but presumably, different combinations of RNA-binding proteins are present in different neurons to determine which Dscam isoforms are produced. Several RNA binding proteins (trans-acting regulators) and RNA sequence elements (cis-acting regulators) are known to be important for Dscam AS; however, little is known about how these factors interact to coordinate specific outcomes. This proposal outlines a research strategy to dissect the mechanisms that control Dscam AS. Aim 1 will focus on dissecting the combinatorial control of Dscam AS in cell culture. Aim 2 will test the requirement for specific RNA-binding proteins in individual neurons. And lastly, Aim 3 will identify common regulators between different neurons that express the same Dscam isoforms. Determining how Dscam AS is controlled in Drosophila neurons will open a window to understanding how animals generate biological complexity and promote proper neuronal function.